Abstract Q fever is a worldwide zoonotic disease that is caused by the obligate intracellular Gram-negative bacterium, Coxiella burnetii. Human Q fever can develop into a severe chronic, potentially fatal disease. Although this organism previously weaponized and currently classified as a category B select agent, no vaccine is commercially available for the prevention of human Q fever in the US. Thus, the creation of a safe and effective vaccine for preventing Q fever remains an important goal for public health and national biosecurity. To achieve this goal, this new R21 application aims to explore the feasibility of designing a multivalent Q fever vaccine targeting novel neutralization-sensitive epitopes of both C. burnetii T cell-independent and T cell- dependent antigens. Despite C. burnetii being an obligate intracellular bacterial pathogen, our recent work demonstrated that formalin-inactivated Nine Mile phase I whole cell vaccine (PIV)-induced protection depends on B cells to produce protective T cell-independent IgM and T cell-dependent IgG. Thus, antigens that activate B cells to produce PIV-specific IgM and IgG are expected to be promising vaccine candidates. Interestingly, our previous studies demonstrated that a peptide mimic of a C. burnetii phase I lipopolysaccharides (PI-LPS) protective epitope (m1E41920) conjugated to keyhole limpet haemocyanin (m1E41920-KLH) conferred significant protection against C. burnetii infection. This finding supports the utility of m1E41920 as a vaccine candidate to prevent human Q fever. However, m1E41920-KLH did not confer the same level of protection as the whole cell vaccine. Thus, the overall objective of this application is to identify additional protective peptide mimics of T cell-independent antigens and PIV-specific protective protein antigens to enhance the protection conferred by this monovalent m1E41920-based vaccine. To achieve this objective, we propose two specific aims to test the central hypothesis that a humoral response to multiple neutralization-sensitive epitopes of both C. burnetii T cell-independent and T cell-dependent antigens is required for conferring the same level of protection as the PIV. Aim 1 will identify additional mimotopes that can mimic multiple protective epitopes on T cell-independent antigens. Aim 2 will identify PIV-specific T cell-dependent IgG recognized protein antigens. This project is significant because it is the critical step towards development of a safe and effective vaccine that can confer an equal level of protection against Q fever as the PIV. Upon the completion of this project, we expect to define the first multivalent Q fever vaccine that targets novel neutralization-sensitive epitopes of both C. burnetii T cell-independent and T cell-dependent antigens.